Rizika

Obecné rizika / NÚ neemových produktů

• Reversible effect on reproduction of both male and female mammals
• Seem to be the most important toxic effects upon sub-acute or chronic exposure [18]

Unprocessed neem products

• Many parts of the neem tree
• Raw plant materials
• Mainly leaves and fruit kernels
• Applied immediately or after drying or grinding
• Toxic components in these materials are not concentrated
• Toxicity is expected to be less pronounced than in extracts
• Accidental ingestion of these materials by grazing cattle or playing children is likely [18]

Aqueous extracts

• Plant extract by soaking of plant material in water
• The effects of aqueous extracts are ambiguous
• Many of the studies do not report dose-effect relations !!!
• Mostly positive effects are mentioned, even after administration of high doses
• But toxic effects were observed at concentrations of 200 mg/kg bw resulting in death of treated goats (Ali, 1987) [18]
• Estimated safe dose (ESD)
• 0.26 and 0.3 mg/kg bw/day
• 2 ul/kg bw/day respectively [18]

Humans

Fermented decoction of neem bark

• Kroes et al. (1993) - Sri Lankan medicine
• Immunomodulatory activity
• In vitro haemolytic assay from healthy volunteers proved inhibited
• Human complement system
• Activity of polymorphonuclear leukocytes

Leaf extract in rats

• Moderate decrease of the blood glucose levels in mice (Mossa, 1985)
• Produced hypoglycaemia in normal rats
• Clotting time of blood was higher than normal
• Serum cholesterol level increased
• Concomitant decrease in liver fat
• Dose-related drop in liver proteins (El Hawary and Kholief, 1990)
• Body weight loss
• Percentage mortality
• Leaf extract was effective against
• Plasmodium yoelii nigeriensis in mice (Obaseki and Jegede Fadunsin, 1986).
• Tail flick reaction time increased
• Reduction in induced writhing was observed in rats
• Naloxone pre-treatment partially reversed the effects
• Effects of the leaf extract were more pronounced than those of the seed oil (Khosla et al., 2000a)

Aqueous leaf extract in mice

• Liver and kidneys were most affected (Ali, 1987) [18]

Reduced

• Tri-iodothyronine (T3)
• Glucose-6-phosphatase activity [18]
• Total testosterone
• Total bilirubin
• Potassium in serum
• Blood glucose levels in diabetic rats (Bajaj and Srinivasan, 1999) [18]

Increased

• Serum thyroxine (T4) concentrations
• Hepatic lipid peroxidation
• Activities of superoxide dismutase
• Catalase activity (Panda and Kar, 2000)
• IgM and IgG concentrations
• Anti-ovalbumin antibody titres
• cholesterol
• urea
• Creatine
• Potassium [18]
• PCV
• Mean corpuscular Hb concentration
• Red blood cell
• White blood cell
• Lymphocyte counts [18]

No influence

• On liver, spleen, thymus or body weight
• No cytotoxic effects were observed (Parshad et al., 1994) [18]

Extrapolace na člověka

• Most relevant NOAEL is 30 mg/kg bw/day with no modulation of the immune responses (Ray et al.,1996)
• Using the standard safety factors
• ADI of 30 × 0.1 × 0.1 = 0.3 mg/kg bw/day can be derived as the safety level of aqueous neem extracts [18]

Azadirachtin

• ESD 15 mg/kg bw/day [18]
• Rats
• Increased serum SGOT and SGPT activities and bilirubin
• Pathological changes in the liver
• Congestion
• Hydropic degeneration
• Necrosis
• Lymphocytic infiltration (Abdel Megeed et al., 2001) [18]
• Subacute azadirachtin increase of:
• The albumin,
• Blood glucose level
• protein content
• White blood cell content
• Platelet counts (Radwan et al., 2001a) [18]
• Azadirachtin during pregnancy (days 6–15)
• No adverse embryo/fetotoxicity and teratogenic effects
• No malformations due to the treatment (Srivastava and Raizada,2001) [18]
• Rats were administered azadirachtin at high dose
• Decrease in
• Body weight gain
• Relative liver weights was observed
• TEC, Hb, erythrocyte sedimentation rate (ESR)
• PCV
• TLC
• Serum protein
• Albumin
• Creatinine
• Increased
• SGOT
• No effect
• Blood urea nitrogen and SGPT
• Histopathologically
• Non-specific generalised degenerative changes were found [18]
• In rats caused
• Increase of the blood urea content
• In uric acid
• Decrease to the normal rate at high concentration (Radwan et al., 2001b) [18]
• No toxicity of azadirachtin in rats
• Even at 5 g/kg bw
• Body weight, vital organs, enzyme activities in liver and serum and blood parameters did not change due to the treatment (Raizada et al., 2001) [18]

Kernel powder

Sheep

• Decrease in the number of nematode eggs in their faeces
• Increase in body weight (Ahmed et al., 1994) [18]
• =antiparazitický efekt - nikoliv toxický

Limonoids nimbolide and nimbinin

• In vitro activity against Plasmodium berghei
• ED50 for nimbolide of 135 mg/kg bw/day (Bray et al., 1985)
• In vivo in mice no anti-malarial activity was observed (Bray et al.,1990) [18]

Neem leaves

• Kadiri et al. (1999)
• Acute toxic effects
• Abdominal upset
• Induce abortion or infertility
• Major features observed
• Oliguria or anuria
• Jaundice
• Anaemia
• Consistent with acute tubular necrosis in all the cases
• Intravascular haemolysis
• Hepatotoxicity
• Direct nephrotoxicity
• 3 out of 53 patients died
• Allergenicity of the neem pollen
• Chakraborty et al. (1998) identified 46 pollen types
• Appeared to be highly allergenic.
• None of these reports mentioned any exposure data,
• Cannot be used to assess the risks [18]

Sheep that ate neem leaves

• Nervous symptoms
• Head movements
• Walkingvin circles
• Dyspnoea
• Increase in body temperature
• Hepatic failure
• Tympanites
• Symptoms lasted for 12 h
• Followed by the death of the animal (Ali and Salih, 1982)

Subacute exposure to leaf powder in male rats

• Decrease in the weight of the seminal vesicle the ventral prostate (Kasturi et al., 1997)
• Reduction in the sperm count and sperm motility
• Increased percentage of malformed sperm (Parveen et al., 1993)
• Dose of 100 mg/rat
• Reduction in the diameters of the seminiferous tubule was observed
• Gradual recovery in histological and biochemical parameters was found after termination of the treatment (Joshi et al., 1996)
• Slightly lower doses
• Height of the epithelium in caput and cauda epididymis was reduced dose-dependently
• Lumen of the caput was packed with lymphocytes
• Serum testosterone concentration was decreased (Kasturi et al., 1995)
• Decreases in
• protein content
• Acid phosphatase activity
• Increases in
• Alkaline phosphatase
• Lactate dehydrogenase (Kasturi et al., 1997)
• Total free sugar
• glycogen
• cholesterol contents (Joshi et al., 1996) [18]

Semi-chronic study

Aladakatti et al. (2001) leaf powder in rats

• Decrease in
• Total sperm-count
• Sperm motility
• Increased
• Relative percentage of abnormal sperm

effects of the powder were annihilated when testosterone was administered simultaneously [18]

• Effects were v.s. due to an androgen deficiency
• Thereby affecting the physiological maturation of sperm

Cattle neem leaf feeding

• Positive effect against intestinal nematodes
• Without any effect on the weight gain (Pietrosemoli et al., 1999)

Rabbits, neem fruits

• Decreased serum activities of
• Acid phosphatase
• Alkaline phosphatase
• Glucose
• Improvement of
• Glutamic oxaloacetate transaminase (SGOT)
• Glutamic pyruvate transaminase (SGPT)
• cholesterol
• Total protein
• bilirubin values (Tanveer et al., 1998) [18]

Extrapolace rizik na člověka

• Effect on protein content of the seminal vesicle is considered to be the most sensitive toxic parameter
• NAEL thus derived can be used for a safety assessment for human consumption
• Using the standard safety factors of 10 for inter- and 10 for intra-species extrapolations
• Results for calculated acceptable daily intake (ADI) for human consumption
• 26.4 × 0.1 × 0.1 = 0.264 mg/kg bw/day
• For 70 kg weighing adult
• 70 × 0.264 mg/kg = 18.5 mg unprocessed leaves/day [18]
• Gives some indication of the range in which a safe dose for daily human exposure to unprocessed neem material i.e. unprocessed leaves could be found [18]

Neem oil nanoemulsion

Lymphocyte toxicity

• Decrease in cellular viability in human lymphocytes
• After 24 hours of exposure in vitro
• At lower concentration (0.7-1 mg/mL)
• Nontoxic while
• At higher concentrations (1.2-2 mg/mL)
• It is toxic
• Oxidative stress induced
• Depletion of catalase, SOD, and GSH levels in human lymphocytes
• Significant increase in DNA damage
• Compared to control in human lymphocytes [6]

Leaf sap

Rat model

• Another study based on rat model
• Administration of leaf sap caused
• Antianxiety effect at low doses
• High doses
• Did not show such types of effect [11] (Jaiswal et al., 1994) [18]

Neemový olej

In adults

• Short-term use of neem
• Is safe
• Long-term use
• May harm the kidneys or liver [1]
• Toxicity associated with neem oil poisoning in an elderly male
• Patient presented with vomiting, seizures, metabolic acidosis, and toxic encephalopathy
• Recovered completely with symptomatic treatment [11]

In small children

• Few cases described
• Neem oil is toxic
• Can lead to death
• Several cases reported neem oil poisoning in children

[18]

• Vomiting
• Hepatic toxicity
• Metabolic acidosis
• Encephalopathy [11]
• Drowsiness,
• Tachypnoea
• Recurrent generalised seizures
• Leukocytosis
• Metabolic acidosis (Lai et al., 1990)
• Sinniah et al. (1982) reported the case of a child, who died
• Changes in the liver and kidneys consistent with Reye’s syndrome / acute aflatoxicosis
• Aflatoxins have been identified in oil samples
• May exist synergistic effects of aflatoxins and other toxic components in the oil [18]

• 5 and 12 ml per young child are clearly toxic
• Safety values for human consumption calculated from animal studies
• much below ± 0.20 ml/kg bw (taking 25 kg for the body weight of a child) [18]

Možné NÚ

• Miscarriages
• Infertility
• Low blood sugar [1]
• Some forms of toxic encephalopathy and ophthalmopathy
• If consumed in large quantities [1]

Podráždění očí nimbovým olejem

• Pozor na obsah silic v oleji
• Nepoužívejte v okolí očí
• Doporučuje se provézt test kožní snášenlivosti [5]

Rabbit model

• Acute toxicity test
• LD50 values of neem oil
• 31.95 mg/kg [11]

Oil can turn rancid

• De Groot, 1991 [18]

Easily contaminated with aflatoxins

• Sinniah et al., 1982 [18]

Acute toxicity of the oil by rats and rabbits

• Dose and time-dependent effects on
• Motor activity
• Respiration
• Orientation within the cage
• Diarrhoea,
• Tremors
• Convulsions
• Medium lethal dose (LD50 value)
• 14.1 ml/kg bw for rats
• 24.0 ml/kg bw for rabbits [18]

Mice and oil exposition

• Not toxic at lower doses
• At high dose
• Hyper-excitability to sound and touch
• Convulsive jerks
• Laboured respiration
• Some animals died (Tandan et al., 1995) [18]

Rats and neem oil

• During the first few days of pregnancy
• Higher abortive effect than later administration.
• 6 ml/kg bw, even 3 out of 13 adult animals died (Lal et al., 1987)
• Oil increased tail flick reaction time
• Reduced induced writhing (Khosla et al., 2000a)
• Normal and hyperglycaemic rats
• Administration of oil caused a lowering of the blood glucose (Dixit et al., 1986) [18]

Subacute effects on female rats

• 2.0–4.6 ml/kg bw
• Oestrous cycle was disturbed
• Reduction in fertility
• Body weight was reduced when the animals were administered a high dose of neem oil (Dhaliwal et al., 1998)

Rabbits and subacute effects

• Lowered blood sugar levels in normal and diabetic rabbits (Khosla et al., 2000b).

Semi-chronic effects

• By Lakshminarayana (1987), Rukmini (1987)
• Debitterised neem oil was found useful as animal feed (Rukmini et al., 1991)
• Three-generation study in male and female rats fed a diet containing debitterised oil did not show any
• Adverse effects on the general health
• Reproductive parameters
• Mean organ weights and the histopathological evaluation of all the organs were similar
• Extract was negative in the Ames test (Chinnasamy et al., 1993) [18]

Úvahy o riziku expozice neemovému oleji

• All together neem oil shows acute toxicity at doses
• 14 and 24 ml/kg bw for rats and rabbits
• If used as an insecticide
• Unlikely that these high levels of intake will be encountered when considering human intake of residues on treated beans
• doses in rats and rabbits would amount to about 1000 ml of oil or more for a 70 kg adult
• Most relevant adverse effect reported in neem-oil exposed animals
• Anti-fertility effect in female rats upon sub-acute exposure
• 2.0–4.6 ml/kg bw (Dhaliwal et al., 1998)
• Lowest observed adverse effect level (LOAEL) for the antifertility effect
• In female rats of 2.0 ml/kg bw
• Safety doses results in a value of 0.2 ml/kg bw
• Extrapolating this to the human results in an ADI of 0.2 × 0.1 × 0.1 ml/kg bw = to 0.002 ml/kg bw
• Implies that a daily intake of 0.14 ml oil for an adult of 70 kg can be considered safe
• This value of 0.002 ml/kg bw is 100 times lower than the dose of 0.2 ml/kg bw reported to be toxic in children [18]

Nimbidin isolated from seeds

• Acute exposure dose-dependently
• Reduced acute paw oedema in rats
• Suppressed induced arthritis
• Supressed fluid exudation in induced granuloma [18]
• Medium effective dose (ED50) 79.4 mg/kg bw in rats (Pillai and Santhakumari,1981)

• Nimbidin to rats, mice and dogs
• Did not produce any signs of toxicity
• Dose-related weight gain
• Increase in Hb level, in liver glycogen
• Reduction in serum protein [18]

• Nimbidin provided a protective effect against ulcers
• In induced gastric and duodenal lesions in rats and guinea pigs
• Enhanced the healing process in acetic acid-induced chronic gastric lesions in rats and dogs (Pillai and Santhakumari, 1984a).

Non-aqueous extracts

• Most toxic neem-based products
• An estimated safe dose (ESD) of 0.002 and 12.5 g/kg bw/day [18]
• Non-aqueous solvents are available
• Extract more apolar, possibly more active constituents
• Often more active than the crude materials they were obtained from
• In most cases analytical characterisation of the extracts is missing
• Impossible to compare the toxicity of the various extracts [18]
• Non-aqueous leave extracts
• Did not show anti-malarial activity in vitro or in vivo in mice (Bray et al., 1990) [18]

Ammonium precipitation

• Isolate active allergenic components from neem pollen
• A skin prick test on human volunteers

A pessary including neem leaf extract

• Did not show any toxicity
• No side-effects
• Was effective in prevention of pregnancy in most of the volunteers (Talwar et al., 1997) [18]

Acetone leaf extract

• Decrease
• Spontaneous activity
• Respiratory rate
• Body and limb tone in mice
• Responses to the environment
• Piloerection
• Dose-dependent hypothermia (Singh et al., 1987)
• CNS depressant activity in mice
• Reduction in locomotor activity
• Reductions in blood pressure and heart rate in rats
• Without showing diuretic activity (Singh et al., 1990)

Methanol extracts of bark and leaves

• Pronounced anti-inflammatory
• Good antipyretic effect in rats and rabbits
• Anti-thrombotic effect in mice (Olajide, 1999)
• Acute oral toxicity in mice
• LD50 cca 13 g/kg bw (Okpanyi and Ezeukwu, 1981) [18]

Ether soluble fraction of alcohol leaf extract

• Analgesic activity in acute inflammatory pain in rats and mice
• Did not show acute toxicity in mice (Tandan et al., 1990).

Petrol ethanol leaf extract

• Anti-inflammatory activity in rats
• Analgesic effect in mice
• Acute toxicity symptoms in mice
• Motor activity, orientation, a reduced reaction to pain and convulsions
• Oral LD50 of the extract was 22 g/kg bw (Koley et al., 1994)

Ethanol leaf extract in mice

• Anti-inflammatory activity
• Inhibition of the proliferative phase of inflammation (Chattopadhyay, 1998)
• Dose-dependently induced mitotic chromosome abnormalities in bone marrow cells of mice [18]
• Appeared even at the lowest dose and remained unchanged in frequency at higher doses
• Increased incidence of structural changes of metaphase chromosomes.
• Probably interfered with DNA
• Chromosome strand breakage
• Or produced spindle disturbances, inducing genotoxic effects (Awasthy et al.,1999).
• Ethanol leaf extract higher than 50 mg/kg bw
• Decreased the blood sugar level
• LD50 value in mice was 4.6 g/kg bw (Chattopadhyay, 1999)
• Ethanol leaf extract
• Did not alter the hepatic glycogen content in normal rats
• In glucose fed rats or in combination with insulin it reduced the hepatic glycogen content (Chattopadhyay et al., 1993a).
• Rodents previously treated with seed extracts
• Complete resorption of embryos on day 15 of pregnancy (Mukherjee et al., 1996a) [18]

Rats on ethanol leaf extract

• Reduced elevated serum levels of cholesterol
• Total lipids and triglycerides (Chattopadhyay, 1995)
• Ethanol leaf extract did not interfere with spermatogenesis (Choudhary et al., 1990) [18]

Hexane seed extract

• Completely abrogated pregnancy
• Restoration of fertility was observed in subsequent cycles
• No further toxic effects were found (Mukherjee et al., 1999) [18]

Evaluation of the data

• The major negative effects of non-aqueous neem extracts
• Effects on male and female reproductive ability
• Singh et al. (1987) found a LOAEL for neuro-psychopharmacologic effects
• 12.5 mg acetone leaf extract/kg bw in mice upon acute exposure
• Extra standard safety factor 10 for extrapolation of the LOAEL to a NAEL
• Calculated safe dose for acute human exposure
• Should be lower than 12.5 × 0.1 × 0.1 × 0.1 = 0.0125 mg of acetone extract/kg bw [18]

Seed cake

• The rest product after oil extraction
• Active ingredients are mostly removed from this material
• Effects on mammals are usually little pronounced (Gangopadhyay et al., 1979; Nath et al., 1989; Ramu et al., 1997) [18]